Reference range of complete blood count, Ret-He, immature reticulocyte fraction, reticulocyte production index in healthy babies aged 1–4 months

Establishing reference ranges of the complete blood count (CBC), reticulocyte hemoglobin content (Ret-He), immature reticulocyte fraction (IRF), and reticulocyte production index (RPI) helps diagnose a disease related to the changes in erythrocyte indices, white blood count, platelets, and reticulocytes, especially in babies. Therefore, the study aims to establish a reference range for CBC and reticulocyte parameters in healthy babies aged 1–4 months. The study design was a cross-sectional study with descriptive analysis of CBC and reticulocyte in babies aged 1–4 months. Three hundred forty-eight babies met the inclusion criteria. This study recruited 89 babies aged 1 month, 87 babies aged 2 months, 86 babies aged 3 months, and 86 babies aged 4 months. The P5-P95 reference range of healthy babies for hemoglobin (Hb) aged 1 month, 2 months, 3 months, and 4 months was 9.95 to 15.45 g/dL, 9.74 to 13.42 g/dL, 9.51 to 12.40 g/dL, and 10.04 to 13.10 g/dL respectively. The P3-P97 reference range of healthy babies for Hb aged 1 month, 2 months, 3 months, and 4 months was 9.60 to 15.90 g/dL, 9.46 to 13.97 g/dL, 9.26 to 12.82 g/dL, and 10.00 to 13.33 g/dL respectively. This study also defined reference ranges for CBC, Ret-He, IRF, and RPI. The reference range of CBC, Ret-He, IRF, and RPI for healthy babies aged 1–4 months in this study can be used as a benchmark.


Discussion
Establishing the lower limit of P3-P97 and P5-P95 on examining specific parameters is commonly used statistically. For example, the WHO (World Health Organization) and CDC (Centers Of Disease Control and Prevention) growth charts set the lower limit of Weight-for-age, Length/height-for-age, Head circumference-for-age, and Arm circumference-for-age at P3 10 . Determination of the lower limit of Hb with P3-P97 and P5-P95, of course, by considering race, ethnicity, age, gender, and altitude above sea level 11 . This study accommodates all reference range limits to be used flexibly.
The results of this study showed the reference range of CBC, reticulocyte count, Ret-He, IRF, and RPI at P3-P97 and P5-P95. Values below the lower limit of the reference range of Hb according to age can be used as a limit to determine if a baby has anemia. In addition, the value below the lower limit of the reference range of Ret-He according to age can be used as a limit to determine whether a baby has ID. The baby is suffered from IDA; if the value of Hb and Ret-He is below the lower limit of the reference range. Thus, for example, this study indicates that the lower limit of Hb for healthy babies aged 4 months (see Tables 1 and 2) can be taken at P3 and P5, i.e., 10.00 g/dL and 10.04 g/dL, respectively. In addition, this study indicates that the lower limit of Ret-He for healthy babies aged 4 months (see Tables 1 and 2) can be taken at P3, and P5, i.e., 17.40 pg and 19.22 pg, respectively. Therefore, to determine whether a baby aged 4 months is suffering from IDA if the value of Hb and Ret-He is below the lower limit of the reference range. Combining several parameters, Hb, MCV, MCH, RDW, reticulocyte count, and Ret He, in conjunction with the peripheral blood smear examination, will make an IDA diagnosis undoubtedly. The examples above can also be used for babies aged 1 month, 2 months, and 3 months (see Tables 1 and 2).
One study showed that IDA, iron depletion and iron deficiency incidence in babies aged 0-6 months were 40.8, 28.0 and 27.0%, respectively. The incidence of IDA in babies aged 0 months, 1 month, and 2 months were 11.8, 10.9 and 11.3%, respectively. This means that the age of 0-6 months, especially 0-2 months, is critical for a baby to suffer from an iron deficiency with or without anemia. Nevertheless, the impact will also affect the incidence of iron deficiency at a later age. Li et al. showed that of 1,127 6-months-old infants in Beijing, the prevalence of anemia was ~ 11.8% 12 . Chen et al. showed that from 509 infants aged 1-12 months, The prevalence of ID and IDA were 3.7 and 2.7%, respectively, in babies under 6 months of age, but increased to 20.4 and 6.6%, respectively, in babies above 6 months of age 13 . Salah et al. show that from 654 infants aged 9-12 months, the prevalence of anemia and IDA was 34.6 and 32.6%, respectively 14 . Nazari et al. found that the prevalence of ID and IDA in children under six years of age was 27.7 and 18.2%, respectively. Considering the high prevalence of ID and IDA in infancy, especially at 0-6 months, iron supplementation in the form of elemental iron at a dose of 1 mg/kg/day should be given to all babies born at term from birth 15 . Another study showed that Daily iron supplementation from early life 36 h at a dose of 2 mg/kg is efficacious for improving iron status and motor development at 6 months in babies at risk 16  www.nature.com/scientificreports/ The results of this study for reference range of other erythrocyte indices, leukocyte, diff count, and platelets can be considered a guide in diagnosing a disease relating to the changes in erythrocyte indices, white blood count, platelets, especially in babies.
Immature reticulocyte fraction indicates the younger fraction of reticulocytes, reflecting erythropoietic activity. Several studies have demonstrated the clinical utility of IRF. For example, an increase in IRF within a few days indicates recovery of bone marrow following bone marrow transplantation, erythropoiesis-stimulating agent therapy, or chemotherapy [17][18][19] . Molina et al. consider an IRF value greater than 10% to indicate early marrow recovery 20 . Chang et al. stated that increased IRF (IRF ≥ 0.23) and increased absolute reticulocyte count (ARC) generally indicated an adequate erythroid response to anemia. Meanwhile, an IRF of 0.23 or less in patients with anemia reflects bone marrow that is nonresponsive or under-responsive to the anemia 21 . Immature reticulocyte fraction may be the first sign of hematologic recovery. It is a powerful indicator of post-chemotherapy aplasia in children with cancer, serving as an additional parameter of impaired bone marrow function 22 . A low total count with a relatively high IRF indicates a regenerating marrow, whereas reticulocytopenia with low IRF is typical of severe aplastic anemia or renal failure. A high total count with high IRF occurs in acute hemolysis and blood loss. In contrast, a low to average total count with a high IRF occurs in dyserythropoietic and during the early response to haematinics. It may also help decide whether macrocytic anemia is megaloblastic or nonmegaloblastic 23 . Mullier et al. showed that IRF, combined with reticulocyte count, may be helpful in hereditary spherocytosis (HS) diagnosis as a high reticulocyte count characterizes HS without an equally elevated IRF. Therefore, a reticulocytes/IRF ratio higher than 7.7 is a precondition for screening HS cases 24 . The IRF increases earlier than the reticulocyte number. It helps monitor the efficacy of therapy in nutritional anemias such as megaloblastic or IDA. Therefore, the reference value of the IRF range in this study can be considered to assess the responsiveness of the anemia therapy given.
The reticulocyte count reflects the erythropoietic activity of bone marrow. In addition, it is valuable in diagnosing anemias and monitoring bone marrow response to therapy. RPI is currently used in pediatrics to assess bone marrow responsiveness to anemia therapy given. However, considering that "conventional" RPI was developed from a study conducted in adults, it is necessary to have an RPI based on standard values adjusted for Table 1. P5-P95 of the white blood count, red blood cell count, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, platelet, red cell distribution with standard deviation, red cell distribution with coefficient of variation, platelet distribution with, mean platelets volume, plateletcrit, neutrophils, lymphocyte, monocyte, eosinophils, eosinophils absolute, basophils, basophils absolute, reticulocyte count, Ret-He, immature reticulocyte fraction, reticulocyte production index, immature platelet fraction, and immature granulocyte for healthy babies aged 1-4 months.  www.nature.com/scientificreports/ different pediatric ages, especially in babies. Therefore, the reference value of the RPI range in this study can be considered to assess the responsiveness of the anemia therapy given. However, according to Bracho et al., the RPI is an inadequate tool for evaluating the bone marrow response in the presence of anemia due to differences in hematologic values between children and adults. In addition, the absence of information on the maturation time of reticulocytes in children 25 . The power of this study is that it parades all reference ranges of CBC, Ret He, IRF, and RPI at P3-P97 and P5-P95 for healthy babies aged 1-4 months. The weakness of this study is that the altitude above sea level, race, genetics, ethnicity, and population of this study is different from other areas. Hence, it certainly affects the reference range of CBC and reticulocyte parameters. In this study, 96.5% reside at altitudes less than 100 m. Again, however, race, genetics, and ethnicity differed from other countries. Therefore, this reference range for CBC and reticulocyte parameters in healthy babies aged 9-11 months can be used in countries with demographic, socioeconomic, and population structures, such as Southeast Asia, especially Indonesia.

Material and methods
Study population. The study design was a cross-sectional study with descriptive analysis of CBC and reticulocyte in babies aged 1-4 months. The study was conducted at 10 Community Health Centers in Banjarbaru, South Kalimantan, from August 2020 to August 2021. Inclusion criteria are babies born at term (gestational age 37-42 weeks) with birth weight ≥ 2500 g, babies are not twins, not taking a hematinic drug, and normal nutritional status. Exclusion criteria were hematological diseases and congenital anomalies. In addition, gestational age and birth weight had obtained from the mother's medical record (Maternal and Child Health Book). The doctor in charge declared the baby healthy. At the time of recruitment, the baby's weight, length, and head circumference were measured by health personnel. Nutritional status is assessed based on body weight and length, divided into good nutrition and undernutrition. Good nutrition if the z-score is − 2 SD to + 3 SD in this study. In contrast, undernutrition if the z-score is < − 2 SD 26 . All of the baby's parents had signed the informed consent. This study obtained ethical clearance from the Research Ethics Commission of the Medical Faculty of the Table 2. P3-P97 of the white blood count, red blood cell count, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, platelet, red cell distribution with standard deviation, red cell distribution with coefficient of variation, platelet distribution with, mean platelets volume, plateletcrit, neutrophils, lymphocyte, monocyte, eosinophils, eosinophils absolute, basophils, basophils absolute, reticulocyte count, Ret-He, immature reticulocyte fraction, reticulocyte production index, immature platelet fraction, and immature granulocyte for healthy babies aged 1-4 months.  Blood sampling. Every baby who meets the inclusion and exclusion criteria will be taken a blood sample of 1 ml from the median cubital vein. First, the blood sample was put in a tube with EDTA anticoagulant, homogenized by turning it over, and stored in a storage box. Then the blood sample was sent to the Banjarbaru Idaman Hospital Laboratory. The Sysmex XN-450 Hematology Analyzer (Sysmex Corporation, Japan) was doing complete blood count and reticulocyte examinations.
Statistical analysis. All baby anthropometry measurements were analyzed by SPSS ver.25 for P3-P97 and P5-P95. Moreover, the CBC and reticulocyte laboratory findings were analyzed by SPSS ver.25 for P3-P97 and P5-P95. All data are presented in narrative and www.nature.com/scientificreports/